It is estimated that, on average, 50% of the 10 million men with diabetes mellitus (DM) have erectile dysfunction (ED) with both Type 1 and Type 2 DM nearly equally associated with ED. Although efficacious orally active PDE5 inhibitors have been developed to treat ED, a large percentage of diabetic men (with estimates as high as 50%) remain refractory to this therapy. The specific hypothesis of this proposal is that an increased corpus cavernosum smooth muscle (CCSM) basal tone and inability to properly relax the CCSM in diabetes-induced ED is mediated by SM calcium sensitization via inhibition of smooth muscle myosin phosphatase (SMMP) activity. Specifically, to address this hypothesis, we will examine the expression and regulation of molecules that either directly or indirectly regulate SMMP activity and the roles that they play in "calcium sensitization/desensitization" pathways will be elucidated. Using CCSM from Type- I and Type II diabetic rats, patients with ED, as well as cultured CCSM cells, our hypothesis will be addressed by accomplishing the following specific aims: 1) To determine, using both Type I and Type II rat models of diabetes a) the precise correlation between diabetes and ED, b) whether there is an increased expression/activity of the "calcium-sensitizing" enzyme Rho-kinase, three well-established ROK regulators (RhoA, endothelin and sphingosine-1-phosphate) and/or the SMMP inhibitory protein CPI-17 in diabetic animals, and the correlation of these changes with ED and c) if there is a simultaneous downregulation of molecules that promote the "calcium desensitization" of smooth muscle (namely PKG-1 and telokin), 2) To determine a) whether similar alterations in the expression/activity of "calcium sensitization/desensitization"- associated molecules that occur in response to experimentally-induced diabetes can be induced in isolated CCSM cells (e.g. high glucose, high insulin, exogenous Et-1, etc.) and b) the ability of pharmacological inhibition of "calcium sensitization" molecules or plasmid-mediated overexpression of "calcium desensitization" molecules to prevent, attenuate or reverse ED and the mechanisms for these effects and 3) To determine, using human CCSM specimens routinely isolated from men undergoing penile surgery to treat ED, whether alterations in the "calcium sensitization/desensitization" pathways in the rat translate to diabetic humans with ED. The data obtained by the completion of the studies described above should provide keen and novel insight into the molecular mechanism for diabetes-induced ED and also establish which "calcium sensitization/desensitization" pathways may serve as attractive molecular therapeutic targets for the treatment of ED. Moreover, since similar changes in "calcium sensitization" pathways are beginning to emerge in diabetic vascular SM, knowledge gained from these studies may have implications in diabetes- induced pathologies beyond the urogenital system.